This invention relates to DNA analysis and, more particularly, to DNA fragment size distribution analysis and sorting. This invention is the result of a contract with the Department of Energy (Contract No. W-7405-ENG-36).
The human genome is comprised of some three billion nucleotides forming the 22 pairs of autosomes plus 2 sex chromosomes, each with continuous DNA pieces of 50-500 million nucleotides. The organization and sequence of DNA forming the human genome contains unique information about the constitution of the organism that provides the DNA. One method for accessing this information is to fragment the DNA at known sites and then to analyze the distribution of fragment sizes, i.e., the number of nucleotides in each fragment between each of the sites. Polymorphisms in the genome structure among individuals lead to substantial variation in the fragment sizes obtained from fragmentation of DNA pieces and allow one to differentiate one person from another or to form a basis for assessing a person's susceptibility to genetic diseases. Analysis of these polymorphisms is often referred to as DNA fingerprinting.
DNA fingerprinting is an important medical diagnostic tool, with additional applications to forensic identification, medical genetics, monitoring the effects of environmental mutagens, basic molecular biology research, and any application that uses gel electrophoresis for DNA fragment sizing and separation. One form of DNA fingerprinting involves "restriction fragment length polymorphism" (RFLP) where restriction enzymes are used to cut a DNA piece from a specific source into shorter pieces, or fragments, of DNA. RFLP provides a unique pattern of DNA fragments containing a unique DNA sequence ordered by fragment size (the DNA fingerprint) when a DNA specimen is digested with restriction enzymes. There are many known restriction enzymes and each recognizes a specific DNA sequence of four to twelve base pairs at which it cuts the DNA, resulting in smaller fragments of DNA.
Once the DNA piece has been cut into many fragments, electrophoresis is conventionally used to separate the fragments by size. An electric field is placed across a gel (either in the form of a slab or packed into a small capillary column) containing the fragments causing the smaller fragments to move faster than the larger ones. Gel electrophoresis is a well known technique and has been used to produce band patterns of DNA fragments that form a fingerprint to identify the individual source of the DNA piece under analysis. The band patterns of specific DNA sequences are conventionally visualized by binding radioactive DNA probes to the separated DNA fragments and exposing suitable film to the radioactive labeled fragments. See, e.g., J. I. Thornton, "DNA Profiling," C&EN, pp. 18-30 (Nov. 20, 1989); K. Heine, "DNA on Trial," Outlook 26:4, pp. 8-14 (1989). In one variation, the fragment ends are tagged with a fluorescent dye so that the fragment migration time along a known path length in an electrophoretic gel can be determined by automated fluorescence detection. See, e.g., A. V. Carrano, "A High-Resolution, Fluorescence-Based, Semiautomated Method for DNA Fingerprinting," 4 Genomics, pp 129-136 (1989).
There are, however, several limitations on the use of gel electrophoresis, particularly where large fragment sizes and radioactive labeling are involved. In both instances, the electrophoretic separation process takes considerable time to provide resolution for large size fragments. The development of images from radioactive probes is an additional time consuming step and has health hazards and environmental concerns associated with radioactive materials. Additionally, the distribution of fragment sizes is logarithmic so that the separation, i.e., resolution between large fragments is less than for small fragments. Electrophoresis also requires relatively large amounts of DNA to obtain a recognizable pattern.
It is desirable to provide a DNA fragment size analysis technique that uses only small quantities of DNA (maybe only a single fragment), provides size information within a short time, and has a high base pair (bp) resolution between fragment sizes. These and other problems of the prior art are addressed by the present invention wherein flow cytometry-based techniques are used to obtain a distribution of DNA fragment sizes from a DNA piece.
Accordingly, it is an object of the present invention to provide rapid determination of DNA fragment sizes.
It is another object of the present invention to obtain a high resolution for sizing DNA fragments, particularly long fragments, i.e., greater than 10 kbp.
One other object of the present invention is to require only a small DNA sample to provide accurate DNA fragment size analysis.
Yet another object of the present invention is to enable fragment length detection without the use of radioactive labels.
A further object of the present invention is to use fluorescent intensities to determine the length of DNA fragments.
Still another object of the present invention is to use the sorting capabilities associated with flow cytometry to sort the fragments by size, i.e., length, for further study.
One other object of the present invention is to obtain an analysis of DNA fragments that is linearly related to the fragment sizes.
Another object of the present invention is to linearly quantitate the number of DNA fragments within any given size class.
It is an object of the present invention to provide an alternative to gel electrophoresis for DNA fragment length sizing.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.